This project seeks to define the mechanism of tumor radiosensitization by manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) gene therapy and optimize conditions for protection of the oral cavity and oropharynx from radiotherapy (RT)-induced mucositis. We have demonstrated in a murine model that intraesophageal, intratracheal or intraoral administration of MnSOD-PL protects normal tissues from single fraction or fractionated irradiation-induced injury. Recent data suggest that there is an antitumor effect of MnSOD overexpression in squamous cancers through depletion of antioxidants, specifically glutathione (GSH). The first specific aim seeks to define the mechanism of MnSOD mediated tumor radiosensitization in cell lines or cell pellets in vitro, and in orthotopic tumors in vivo. We will determine whether there is a positive or negative effect of adding the antitumor agent, epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) ZD1839 (Iressa), to MnSOD-PL. Novel real time in situ measurement of peroxynitrite and H2O2production in irradiated (and/or Iressa-treated) orthotopic tumors compared to normal tissue will be used to quantitate directly redox changes during MnSOD-PL tumor radiosensitization. In the second specific aim, using normal mice treated with single fraction or fractionated RT (and/or Iressa), we will determine the optimal schedule and dose of intraoral delivery of MnSOD-PL and quantitate several objective parameters of RT-induced damage including mucositis, ulceration of the tongue, saliva output, osteoradionecrosis, and fibrosis. The third specific aim seeks to determine the schedule and dose of intravenous (I. V.) and/or intraoral MnSOD-PL required for protection from irradiation mucositis in two rodent squamous cell carcinoma models of orthotopic tumors in C3H/HeNHsd and nude mice, respectively, and determine effect on the antitumor response. Using an epitope hemagglutinin (HA)-tagged MnSOD transgene, the depth of penetration of transgene expression in cells of an orthotopic tumor compared to cells of the oral cavity, including the floor of the mouth, hypopharynx, oropharynx, mobile and base of the tongue, as well as major and minor salivary glands, will be quantitated. We will determine whether I.V. MnSOD-PL enhances orthotopic tumor killing by irradiation plus Iressa and relate this quantitatively to normal tissue and tumor antioxidant levels. This project should validate the use of radioprotective gene therapy for improving the quality of life in head and neck cancer patients, and may also facilitate irradiation dose escalation by modulating side effects.